Millions of people in the U.S. suffer from hearing loss caused by permanent damage to sensory hair cells of the inner ear. Hair cell damage often results from exposure to excessive sound or particular licit drugs, such as the aminoglycoside antibiotics. The overall goal of this proposed research is to discover novel drugs that prevent aminoglycoside-induced hair cell death and preserve hearing loss in patients requiring these life-saving antibiotics. For this work we will use the zebrafish lateral line, which is a powerful n vivo model for studying mechanisms of hair cell death and protection. Our preliminary data indicate that caffeine significantly protects hair cells from aminoglycoside damage. The specific goal of this research is to determine the mechanism by which caffeine confers protection, and to identify additional natural products that prevent aminoglycoside-induced hair cell death. This project has two specific aims: 1) Test the hypothesis that caffeine otoprotection is mediated by inhibition of p53 and adenosine signaling, and 2) Test the hypothesis that natural products are novel agents for preventing aminoglycoside ototoxicity. Our previous research demonstrates that aminoglycosides activate p53 in damaged hair cells. Caffeine is a known p53 inhibitor, in part via its interaction with adenosine signaling. For Aim 1, we will use pharmacologic and genetic manipulation to modulate p53 and adenosine receptors during aminoglycoside treatment. Caffeine is a plant alkaloid and other natural products, including plant and microbial extracts, likely represent an untapped source of novel otoprotective agents. In Aim 2, we will conduct a series of drug discovery experiments to identify natural products that act as hair cell protectants and to characterize their mechanisms of action. These studies will provide new lead compounds for auditory drug development. By specifically targeting caffeine, a widely used compound with a well-characterized safety profile, we increase the likelihood that our findings may be quickly translated into clinical application. Student researchers are integral to all aspect of this project. Students will learn cutting- edge cell biology and drug discovery techniques that will bolster their biomedical career opportunities, while actively contributing to the discovery of new compounds that prevent drug-induced hearing loss.